Observations of Infection Structures after Inoculation with Colletotrichum orbiculare on the Leaves of Cucumber Plants Pre-inoculated with Two Bacterial Strains Pseudomonas putida or Micrococcus luteus

Infection structures were observed at the penetration sites on the leaves of cucumber plants inoculated with Colletotrichum orbiculare using a fluorescence microscope. The cucumber plants were previously drenched with suspension of bacterial strains Pseudomonas putida or Micrococcus luteus. The plants pre-inoculated with both bacterial strains were resistant against anthracnose after inoculation with C. orbiculare. To investigate the resistance mechanism by both bacterial strains, the surface of infected leaves was observed at the different time after challenge inoculation. At 3 days after inoculation there were no differences in the germination and appressorium formation of conidia of C. orbiculare as well as in the callose formation of the plants between both bacteria pre-inoculated and non-treated. At 5 days, the germination and appressorium formation of the fungal conidia were, however, significantly decreased on the leaves of plants pre-inoculated with M. luteus at the concentration with 1.0 x 10(7) cfu/ml. Furthermore, callose formation of plants cells at the penetration sites was apparently increased. In contrast, there were no defense reactions of the plants at the concentration with 1.0 x 10(6) cfu/ml of M. luteus. Similarly, inoculation P. putida caused no plant resistance at the low concentration, whereas increase of callose formation was observed at the higher concentration. The results of this study suggest that the resistant mechanisms might be differently expressed by the concentration of pre-treatment with bacterial suspension.

Selection and Efficacy of Soil Bacteria Inducing Systemic Resistance Against Colletotrichum orbiculare on Cucumber

Soil bacteria were screened for the ability to control cucumber anthracnose caused by Colletotrichum orbiculare through induced systemic resistance (ISR). Sixty-four bacterial strains having in vitro antifungal activity were used for selecting ISR-inducing strains in cucumber. Cucumber seeds (cv. Baeknokdadagi) were sown in potting mixtures incorporated with the soil bacteria, at a rate of ca. 10(8) cells per gram of the mixture. Two week-old plants were then transplanted into the steam-sterilized soil. Three leaf-stage plants were inoculated with a conidial suspension (5x10(5) conidia/ml) of C. orbiculare. Diseased leaf area (%) and number of lesions per cm2 leaf were evaluated on third leaves of the plants, 5~6 days after inoculation. Among 64 strains tested, nine strains, GC-B19, GC-B35, GK-B18, MM-B22, PK-B14, RC-B41, RC-B64, RC-B65, and RC-B77 significantly (P = 0.05) reduced anthracnose disease compared to the untreated control. In contrast, some bacterial strains promoted susceptibility of cucumber to the disease. From the repeated experiments using the nine bacterial strains, GC-B19, MM-B22, PK-B14, and RC-B65 significantly (P = 0.05) reduced both diseased leaf area (%) and number of lesions per cm2 leaf in at lease one experiment. These strains with control efficacy of 37~80% were determined to be effective ISR-inducing strains.

Different Mechanisms of Induced Systemic Resistance and Systemic Acquired Resistance Against Colletotrichum orbiculare on the Leaves of Cucumber Plants

Defense mechanisms against anthracnose disease caused by Colletotrichum orbiculare on the leaf surface of cucumber plants after pre-treatment with plant growth promoting rhizobacteria(PGPR), amino salicylic acid(ASA) or C. orbiculare were compared using a fluorescence microscope. Induced systemic resistance was mediated by the pre-inoculation in the root system with PGPR strain Bacillus amylolquefaciens EXTN-1 that showed direct antifungal activity to C. gloeosporioides and C. orbiculare. Also, systemic acquired resistance was triggered by the pre-treatments on the bottom leaves with amino salicylic acid or conidial suspension of C. orbiculare. The protection values on the leaves expressing SAR were higher compared to those expressing ISR. After pre-inoculation with PGPR strains no change of the plants was found in phenotype, while necrosis or hypersensitive reaction(HR) was observed on the leaves of plants pre-treated with ASA or the pathogen. After challenge inoculation, inhibition of fungal growth was observed on the leaves expressing both ISR and SAR. HR was frequently observed at the penetration sites of both resistance-expressing leaves. Appressorium formation was dramatically reduced on the leaves of plants pre-treated with ASA, whereas EXTN-1 did not suppress the appressorium formation. ASA also more strongly inhibited the conidial germination than EXTN-1. Conversely, EXTN-1 significantly increased the frequency of callose formation at the penetration sites, but ASA did not. The defense mechanisms induced by C. orbiculare were similar to those by ASA. Based on these results it is suggested that resistance mechanisms on the leaf surface was different between on the cucumber leaves expressing ISR and SAR, resulting in the different protection values.